Voltage gated proton channels are proteins found in many cell membranes that allow protons to cross the membrane in a highly regulated manner. For over two decades, these molecules were studied in cells using electrical measurements (voltage-clamp). The identification in 2006 of the gene that codes for these molecules makes it possible to study the molecular structure in unprecedented detail. In this project, specific mutations in the proton channel gene will be produced and the mutant channels will be expressed in cell lines. The effects these mutations have on molecular behavior will be evaluated using patch-clamp measurements. One goal is to determine how proton channels sense pHo and pHi, the proton concentrations on either side of the cell membrane, which is crucial to their ability to carry out their functions. We hypothesize that one or a few amino acid residues that are accessible to the solution bind protons and thus act as pH sensors. The second goal is to determine which parts of the molecule sense membrane potential. Because the proton channel molecule resembles the voltage-sensing domains of other ion channels, the first attempt will be to determine if the same residues sense voltage. Next, other charged amino acids located in membrane-spanning regions will be examined. Third, the location of the pathway by which protons pass through the molecule will be determined. The first hypothesis tested will be based on similarities to corresponding domains of other channels. The unique aspect of proton channels is a conduction mechanism of proton hopping (Grotthuss-like) that could occur at a titratable reside. Next the accessibility to external or internal solutions of amino acid residues at specific locations in the molecule will be examined systematically, by inserting cysteine residues at specific locations and then determining the rate at which they can be modified chemically (cysteine scanning). Finally, the discovery in 2008 that proton channel is a homodimer raises questions that will be addressed. We will test whether the inhibitor Zn2+ binds with high affinity between the two monomers. These and other experiments will indicate whether the two monomers function independently, or if they interact.